Magnetic nanoparticles coupled to annexine, and utilization thereof

ABSTRACT

The invention relates to a process for producing ferrofluid-forming magnetic particles substituted by DMSA (FFSH) that can be covalently coupled, directly or through a difunctional reactant, to an effector, which process comprises combining DMSA with ferrofluid-forming magnetic particles to form FFSS particles having disulfide bonds, and reducing the disulfide bonds of the FFSS at basic pH to form FFSH. The invention includes a composition comprising such FFSH magnetic particles. The invention also relates to a method in which FFSH magnetic particles are incubated with an effector comprising at least one functional group which forms an S--S, C--S, C--C, or C--N bond with DMSA, so that a covalent effector-FFSH complex is formed. The invention further includes a method for separating and/or distinguishing between molecules or cells having a ligand that forms an affinity complex with an effector-FFSH particle of the invention, and those molecules or cells that do not have such a ligand.

FIELD OF THE INVENTION

The present invention relates to a new means making it possible todifferentiate and/or separate components of a complex mixture,especially cells, still more particularly red corpuscles, and exhibitingat their surface a receptor specific for an effector coupled to magneticparticles. The surface complexing of the particles by dimercaptosuccinicacid (DMSA) of formula HOOC--CHSH--CHSH--COOH makes it possible toobtain a ferrofluid which is stable in aqueous medium. The inventionalso relates to an improvement to the process for preparation of theseDMSA-treated ferrofluids, making the latter capable of being employed ina process of differentiation or of separation of cells or of molecules.

In what follows, the term "effector" or "ligand" means any molecule ormacromolecule that is free or bonded to a structure which contains it orwhich carries it, and capable of directly or indirectly forming anaffinity complex with another molecule or macromolecule.

Also in what follows the numbers within brackets refer to the literaturereferences at the end of the description.

BACKGROUND OF THE INVENTION

Biomedical applications of colloidal solutions of magnetic particles, orferrofluids, have developed essentially in three directions:

imaging (MRI) as contrast agents (1, 2, 3),

magnetic separation of various cells, organites or biological molecules(4, 5, 6, 7, 8, 9)

destruction of target cells by creation of a local hyperthermia underpulsating magnetic field (10).

The most extensively employed magnetic particles are the ferrites MFe₂O₄ (including magnetite Fe₃ O₄) and maghemite γ Fe₂ O₃. The surface ofthe particles must be conditioned in order to obtain colloidal solutionsthat are stable in physiological medium. In most cases the particles arecoated with macromolecules such as carbohydrates like dextran (10, 12,1), proteins like albumin (5, 8) or synthetic polymers likemethacrylates and organosilanes (7, 9, 13). However, as Gromanindicates, covering the particles with macromolecules of high molecularmass does not make it possible to obtain sols that are stable in thelong term. The macromolecules separate from the particles, which thengradually aggregate. Other methods of conditioning the surface of theparticles have been proposed, such as the use of molecules of lowmolecular mass, containing complexing groups such as phosphates,phosponates and carboxylates (2). Very particularly, hydroxylatedpolycarboxylic acids such as citric and tartaric acids andpolycarboxylic acids containing thiol groups, like dimercaptosuccinicacid (DMSA) are complexed with the surface atoms of iron(III) (14). Eachof these complexing molecules is bonded to one or more surface sites ofthe particles. The aqueous sols thus obtained are very stable inphysiological conditions.

In many biomedical applications of the magnetic particles the lattermust be coupled to a specific protein. Thus, in the case of cell sortingunder magnetic field the particles must be capable of bondingspecifically to the target cells. This recognition is often ensured bythe formation of an antigen-antibody complex between a surface antigenof the target cell and an antibody bonded to the particles. The proteinmay be either adsorbed directly at the surface of the particles (4) orbonded covalently (5, 7, 13). The use of difunctional intermediatecompounds such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP)(8, 14) makes it possible to bond an antibody strongly to a particle viaa peptide bond and a disulfide bridge, without damage to the protein orto its antigenic properties (15).

French patent No. 2 662 539 (14) described a process for obtainingfinely divided magnetic supports by controlled modification of particlesfilled with precursor ferrofluid, including a stage of treatment with anagent capable of modifying the nature of the surface of the saidparticles, and so as to make them stable in polar or nonpolar solventsin wide pH ranges and to endow them with a chemical or biologicalreactivity. An example of such a modification is a DMSA-based ferrofluidmaking it possible to render thiol groups available and reactive at thesurface of these so-called substituted ferrofluids; however, in theusual pH and ionic strength conditions, these thiol groups tend tobecome oxidized and to form disulfide bridges making these ferrofluidsill-suited to a use as constituents of a magnetic effector.

A use, especially for differentiating and/or separating cells which haveat their surface a receptor for a ligand signaling a property or aparticular physiological state, made it necessary to improve theferrofluids of patent 2 662 539 (14) in such a way that disulfidebridges do not limit the coupling; Such an improvement then making itpossible to produce DMSA ferrofluid complexes coupled covalently to thesaid effectors and then to employ them as means for differentiating andseparating complexes or cells carrying, if appropriate, the receptor forthe said effector.

The effector/receptor affinity pairs are numerous and the invention asdescribed below will be capable of being easily applied by a personskilled in the art to the pairs of receptors of interest to him/her, aswell as to other affinity pairs such as antibody/antigen,lectin/polysaccharide, biotin/avidin and nucleic acid (+ and - strands)pairs and the like, as soon as the effector can be coupled to theparticles through the intermediacy of a difunctional reactant optionallycomprising thiol residues one of the functional groups of which permitsthe bonding to the effector and the other functional group of which iscapable of forming S--S, C--S, C--C or C--N bonds with DMSA.

A particular application of the use of the ferrofluid coupled to aneffector is the use as effector of an annexine, like annexine V, whichhas a particular affinity, in the presence of calcium ions, for anionicphospholipids, like phosphatidylserine (PS) and in, a lesser degree,phosphatidylethanolamine (PE). These compounds of the plasmic membranesare essentially localized in the inner layer of the membranes, incontrast to other phosphatidylcholine and sphingo-myelin type compoundswhich are, on the contrary, predominantly localized in the outer layerof the plasmic membranes. The cell membranes exhibit an asymmetry suchthat, when the membrane is healthy, the phosphatidylserines and some ofthe phosphatidyl-ethanolemines residing on the inner layer areinaccessible, whereas, when the membranes are perturbed, there is arandom localization of these same phospholipids. Consequently, thosethat are localized in the inner layer of the membranes become accessibleover the whole cells and, in particular, phosphatidyl-serine becomesaccessible to a coupling by an annexine like annexine V or capable ofbeing attacked by an external phospholipase. This perturbation may bedue to an inflammation, to an apoptosis (16), to autoimmune reactions(17), to a sickle cell anemia (18, 19), or to a pathological orinfectious state of the individual (19, 22), or simply to an excessiveaging of the cells and especially of the blood cells during theirstorage in vitro (23). It is thought that the presence ofphosphatidylserine in the outer layer of the plasmatic membraneconstitutes a signal for the elimination of the cell by the immunesystem (24, 25).

Patent application WO 91/09628 relates to the use of anticoagulantpolypeptides of the annexine class which are provided with a marker andthese marked annexines or VAC (vascular anticoagulant protein) areemployed as means for differentiating phosphatidyl-serines fromphosphatidylcholines and for thus diagnosing a prethrombotic state viathe presence of a coupling of annexine to the cells exhibiting aphosphatidylserine in an accessible manner.

Annexines are a group of homologous proteins, from 35 to 45 k daltons,which are found in all mammals at different stages of development, aswell as in other vertebrates such as arthropods, slime molds (26),yeasts, sponges, fungi, protozoa, plants and bacteria (27, 28). Allmammalian cells, with the exception of erythrocytes, produce an annexinelike annexine V. The structure of annexines and their properties aredescribed in reviews (28, 29). These proteins have been sequenced andsome of them are available as recombinant proteins (27). As set outabove, cell membranes which have been damaged by a mechanism of somekind exhibit an increased capacity for binding annexines; this can beobserved, for example, by comparing fresh erythrocytes with erythrocyteswhich have been subjected to a relatively long storage period. Insofaras this increase in capacity for bonding to annexine is the reflectionof an anomaly, the measurement of the erythrocytes' capacity forcoupling to annexine is found to be an important tool for the control ofquality of the blood to be employed in blood transfusions.

Selection of blood for transfusion is done essentially using threecriteria: the immunological compatibility of the donor and of thereceiver, the absence of viral or parasitic contamination, and theabsence of pathology in the donor. Suitable tests have been developedfor verifying blood groups and possible viral or parasiticcontaminations; the donor's state of health is evaluated simply byinterrogation. It is of interest, however, to note that there is nothingin existence for controlling the degradation of the blood during itspreservation, it being known that individual bloods can age at differentrates. Another potential problem which can arise in the selection of theblood for transfusion is that of its control when a number of viruses orof pathogenic agents are still unknown and hence undetectable.

BRIEF SUMMARY OF THE INVENTION

A means, as proposed in the present invention, which has the advantageof being simple in use, makes it possible to discriminate the cellscapable of coupling to annexines, in particular to annexine V, in thepresence of calcium and exhibiting a perturbed membrane state, fromnormal cells in the mixture, and to determine their significance.

The present invention is an improved process for obtaining magneticparticles complexed with DMSA, forming ferrofluids called FFSH, the saidferrofluids being capable of being directly or indirectly coupled viacovalent bonds to an effector making it possible to form FFSH-effectorcovalent complexes, the said process including the following additionalstages in relation to the process described in French patent 2 662 539:

a) the FFSS, which consist of particles associated with the oxidationproducts of DMSA, are centrifuged before peptization of the flocculateformed when the ligand is added,

b) the FFSS are next reduced by addition of dithiothreitol (DTT), whichcuts the disulfide bridges and regenerates the DMSA, at basic pH, toform FFSH, c) the excess DTT is removed by flocculation of the FFSH atpH 2, the FFSH retained on a magnet being subsequently washed andresuspended in a neutral buffer, for example a PBS buffer.

The invention also relates to the process for obtaining DMSA-substitutedmagnetic particles (FFD) coupled covalently to an effector, and whichincludes the following stages:

a) Obtaining effectors exhibiting accessible thiol groups:

For the effector to be able to form covalent bonds with the thiol groupsof the FFSS or of the FFSH, it must itself comprise thiol groups. Eitherthe macromolecule itself comprises these said residues, or they aregenerated covalently by coupling to a difunctional reactant, whichreactant itself must have these residues capable of reacting with the SHgroups of the DMSA and a second reactive functional group capable offorming covalent bonds with the effector itself. A difunctional reactantwhich could be employed in this context is SPDP, which, besides a bondwith a thiol group, is capable of forming a bond with primary amines;however, it is also possible to mention N-succinimidylS-acetylthioacetate (SATA), succinimidyl bromoacetate or carboxydiimide,maleimides and the alkyl or aryl sulfonyl (tresyl, tosyl, and the like)chlorine-containing derivatives. The difunctional reactant, for exampleSPDP, is added to the effector in an anhydrous solvent (for example1-methyl-2-pyrrolidone) and incubated for 2 hours at ambienttemperature. The excess SPDP is removed by filtration or gel or bydialysis.

b) Coupling of the effector to FFSH: The effector exhibiting groups thatcan act with thiols either directly or indirectly by coupling with thedifunctional reactant in the conditions described above is incubated inthe presence of ferrofluids treated with DMSA and is reduced with DTT(FFSH) according to the process described above, thus forming aneffector-FFSH covalent complex; a covalent complex is intended to meanthat the covalency resides in the bond at the thiol groups, by formationof S--S bridges between the SH residues of the DMSA and the SH orsulfonic ester residues of the effector which is natural or substitutedwith the difunctional reactant; it may happen that thiol or other groupsof the FFSH have not reacted in the preceding stages and, whereapplicable, these groups are masked by a saturation treatment with serumalbumin which, after formation of the effector-FFSH complex (or thosebonding the effector to the particles via another difunctional agent)makes them incapable of reacting with another reactant.

The effector-FFSH covalent complexes obtained (or those bonding theeffector to the particles via another difunctional agent) are thuscapable of forming affinity complexes with receptors or halves ofaffinity pairs capable of coupling to the effector, and the magneticproperty of these complexes can be employed for discriminating,measuring and/or separating the effectors-FFSH which have reacted fromthose which have not reacted.

Such a separation is particularly advantageous when it is desired toseparate categories of cells which differ in the nature of a receptor atthe surface of the latter; this is because the existing separationmethods are often traumatizing for the cells and the separations whichcan be applied to macromolecules, especially affinity chromatographs,are of little practical use for cell separations. A person skilled inthe art knows in which case it is particularly advantageous to separatea particular category of cells in a complex mixture; the most strikingexamples which may be mentioned are the bone marrow strain cells fortreating ex vivo and reinjecting them into patients in order to treatthem using gene therapy, or else some categories of lymphocytes, toharvest the platelets which have not been activated and, more generally,any cells which it is desired to separate or eliminate, for examplefollowing a viral infection in order subsequently to reinject the cell"pool" in a homologous or autologous manner into individuals.

With this in mind, there is a category of cells which it is extremelydifficult to discriminate: these are erythrocytes or red corpuscleswhich originate from ill or infected individuals or else which havedegraded following an excessively long conservation period in vitro. Thecontrol of quality of the red corpuscles or of corpuscle concentratestoday remains a major problem of transfusion therapeutics, with a doubtcontinuing to exist with regard to the health quality of the redcorpuscles which are injected into patients, insofar as an infectionthat is too recent cannot be detected by conventional means ofdiagnostics, as it is not possible to detect infections by unknownpathogens, and as it is not possible to allow oneself to prolong thestorage of pouches of blood without the risk of damaging a certainnumber of blood cells.

It was stated above that the cell membranes and especially the cellmembranes of the red corpuscles are damaged or perturbed as a result ofvarious pathologies or of their aging, or even of interrupting thecooling line, such a perturbation giving the cells an ability to bond toannexines.

The invention also relates to a process for obtaining magnetic particlesforming ferrofluids complexed with DMSA so as to be able to be coupledto an annexine. Thiol groups can be generated by covalent coupling ofannexines to SPDP. In the process of the invention and in order to formFFSH-annexine complexes, called AnxFF, the relation between the FFDTTand the annexine-SPDP is between 30 and 120 μg and preferably between 30and 60 μg per 100 ml of FFSH.

The invention also relates to ferrofluid particles complexed with DMSAand treated with DTT by a process as described above, and to ferrofluidparticles coupled covalently through the intermediacy of disulfidebridges between the DMSA and the effectors, which complexes are obtainedby the process described above. A preferred embodiment of thesecomplexes is ferrofluid particles coupled to an annexine.

In what precedes and what follows we shall speak without discriminationof effector-FF and effector-FFSH particles, of FF or FFD-effectorcomplexes and when the effector is an annexine or an active peptidederived from annexine like, for example, the N-acetyl-lipocortine ipeptide derived from annexine I (30), of Anx FF; and in all cases theyare complexed with DMSA and then treated with DTT according to theimprovement of the invention and coupled with a difunctional agent.

The present invention also relates to the use of the effector-FFSHcomplexes obtained by the process described above, for thedifferentiation and/or for the separation of the compounds carrying areceptor for the said effector from those not carrying the saidreceptor. The receptor here is understood to mean a molecule or amacromolecule which can form an affinity complex with the effector,regardless of whether this molecule or macromolecule is free in a mediumor incorporated into a complex structure, for example a membrane.

In a preferred use according to the invention the ligand is an annexineand the receptor of the ligand is phosphatidylserine (PS) orphosphatidyl-ethanolamine (PE), phospholipids capable, in the presenceof Ca²⁺, of bonding to the Anx FF complex after perturbation of themembrane structures; in this case the use according to the inventionmakes it possible to differentiate and/or to separate the cellsexhibiting a state which is abnormal--and hence capable of bonding toAnx FF in the presence of calcium ions--from the healthy cells incapableof binding these particles.

In the use of the invention the particles of the Anx FF are between 3and 30 nm and preferably between 5 and 15 nm in size.

As stated above, the control of the erythrocytes before a bloodtransfusion or an injection of a corpuscle residue continues to presenta problem of health safety of the said cells. One use of the particlesaccording to the invention makes it possible to palliate the deficiencywhich existed hitherto: in a whole blood or in a corpuscle concentrateit is today possible to envisage detecting the presence of erythrocytesexhibiting an abnormal physiological state due to an excessive aging orbeing the reflection of a person's pathological state and, according towhat is desired, to separate these abnormal cells from the healthy cellsor simply to check a sample taken before a possible transfusion; theparticles of the invention can, finally, be employed in a process ofidentification of a possible pathological state, as a preliminary stagebefore a more advanced identification by other means which are known toa person skilled in the art; this may be, for example, the presence ofan inflammatory reaction, of autoimmune reactions or of a perturbationin the coagulation sequence. The annexine-ferrofluid (Anx FF) complexescan also be employed for a control of quality of the lymphocytes and ofthe platelets before these cells are employed, for example, fortherapeutic purposes.

Recent observations (22) have shown that nitric oxide synthetase isinhibited by the phosphatidylserines secreted or exposed at the surfaceof the cells; in this context, the Anx FF particles of the invention canbe employed for the manufacture of a medication intended for treatingthe immunity deficiencies induced by viruses or parasites.

The invention also relates to a method for differentiating and/orseparating cells exhibiting an abnormal physiological or pathologicalstate manifesting itself in the presence of a receptor for annexine,especially phosphatidylserine, at their surface, the said methodincluding:

a) coupling of the Anx FF particles, obtained by a process describedabove and detailed in the examples below, to the cells in the presenceof calcium ions;

b) separation of the cells coupled to the Anx FF from the uncoupledcells by the use of a magnetic field,

c) counting or evaluation of the cells which have coupled to the Anx FF,

d) the evaluation of the proportion of the healthy cells being carriedout by the measurement of the relationship between the cells which havenot reacted with the Anx FF and the total number of cells.

This method of the invention is applied preferably to erythrocytes; inthis method the calcium ions are provided by CaCl₂ in a concentration ofbetween 0.5 and 5 mM.

The core of this device for differentiation and/or separation is themagnetic property of the Anx FF particles; the invention also relates toa device permitting this differentiation and/or this separation, whichwould employ this property; the device according to the inventionincludes at least:

a) an electromagnet,

b) a pump,

c) a means for delivering the sample,

d) a means for delivering the buffer containing Ca⁺⁺ ions,

e) a means for delivering the washing buffer,

f) valves making it possible to control the flows of the differentfluids mentioned in c), d) and e).

The examples below and the various figures illustrate the inventionwithout, however, any limitation thereof being implied.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: automatic device for sorting red corpuscles or another targetadopted using Anx FF.

FIG. 2: influence of the Ca²⁺ cations on the coupling of Anx FF witherythrocytes.

FIG. 3: change in the coupling of erythrocytes with Anx FF in the courseof storage in vitro.

FIG. 4: degree of retention of the erythrocytes by Anx FF as a functionof the number of cells employed in the test.

FIG. 5: degree of retention of the erythrocytes as a function of thequantity of Anx FF employed in the test.

DETAILED DESCRIPTION OF THE INVENTION Example 1: Preparation of the DMSAFerrofluids (FFSH)

The starting ferrofluid is a nitric ferrofluid synthesized according tothe method of French patent 2 662 539. It consists of nanoparticles ofmaghemite γFe₂ O₃ of a diameter of between 3 and 30 nm (mean diameter: 9nm) charged positively at the surface in acidic medium (pH<6) ornegatively in basic medium (pH>10).

The preparation of a sol which is stable at about pH 7-8 for producing acoupling in a homogenous liquid phase in conditions which are notdenaturing for the proteins (in this case annexine) or other biologicalmolecules is done by complexing of the surface irons using DMSA. Thefree thiol groups permit the formation of S--S bond with the SPDP boundbeforehand to the protein via a peptide bond.

1st stage: 5 cm³ of nitric ferrofluid ([Fe] =1 mol 1⁻¹) are added to 250cm³ of a 10⁻³ mol 1⁻¹ solution of DMSA in degassed distilled water. The[DMSA]/[Fe] ratio is 0.05. Flocculation takes place and after 30 minutesthe pH is near 4.5.

2nd stage: the excess DMSA is removed by centrifuging: 10 minutes at3000 rev/min.

3rd stage: the flocculate is peptized with a 0.1 mol 1⁻¹ sodiumhydroxide solution to pH 11. A sol stable between pH 3 and 11 is formed.The volume is adjusted to 50 cm³ and the pH to 7 with a dilute HClsolution. This DMSA-ferrofluid ([Fe] =10⁻¹ mol 1⁻¹) is stable in timeand serves as master solution for its subsequent applications.

4th stage: before coupling to the protein-SPDP, 1 cm³ of the aboveferrofluid is treated by the addition of 1 cm³ of an aqueous solution ofDTT at a concentration of 10⁻² mol 1⁻¹ at about pH 9. It is left toincubate for 1 hour and the mixture is then flocculated by the additionof 0.1 mol 1⁻¹ HCl to pH=2. After centrifuging for 10 minutes at 5000rev/min the flocculate is peptized with a PBS solution (0.1 mol 1⁻¹) andthe pH is adjusted to 7.4. This precipitation-peptization sequence isrecommenced twice in order to remove completely the excess DTT and theDMSA possibly released during the cutting of the disulfide bridges.During the last peptization the volume of PBS is adapted to the desiredconcentration for the ferrofluid before its coupling by directincubation with the protein-SPDP.

Example 2: Preparation of the Annexine-Ferrofluid Complex

1) The ferrofluid is treated with dimercaptosuccinic acid (DMSA) andreduced with dithiothreitol at pH 9.2 and at ordinary temperature(FFSH). After flocculation of FFSH at pH 2 and centrifuging, thesupernatant containing the excess DTT is removed and the flocculateretained by a magnet is washed and then dispersed in a phosphate buffer(PBS). The thiol groups of the FFSH are determined using Ellman'sreagent (5,5'-dithiobis(2-nitrobenzoic acid) (31), with minormodifications to remove the interference by the ferrofluid particles inthe spectrophotometer reading.

2) An annexine, in this case recombinant annexine V, is reacted withSPDP in PBS medium, the SPDP being in excess in relation to the annexine(2.5 moles of SPDP per mole of annexine). The SPDP is dissolved in ananhydrous solvent, 1-methyl-2-pyrrolidone, and then added to theannexine in an aqueous medium; the mixture is left to incubate atambient temperature. The annexine-SPDP is obtained and the excess SPDPis hydrolyzed spontaneously.

3) Various quantities of annexine SPDP are incubated with the FFSH for 2hours at ambient temperature, the optimum ratio of the two componentsbeing determined experimentally, for example by measuring the change inthe electrophoretic mobility of the particles in solution by employingthe Doppler effect. In this case the electrophoretic mobility of the AnxFF complex is compared with that of the FFSH; another method is thedetermination of the enzymatic activity or of another functionalproperty of the effector, such as its affinity for an antibody, or theability to form an affinity complex with antibodies of aperoxidase/protein A complex immobilized on the particles, and thedetermination of the peroxidase activity. An optimum activity isobtained by the binding of 1.5 to 2 μg of annexine on 1 ml of FFSH, asthe use of the above-mentioned methods has shown.

4) The DMSA which has not reacted with the effector and otherpotentially reactive sites on the ferrofluid particles are masked bysaturation with 1% of BSA (for bovine serum albumin) in 50 mM of NaHCO₃buffer and pH 9.6 to give the Anx FF. In some cases other proteins suchas human serum albumin may be more appropriate. A particular attentionis paid to avoid a flocculation resulting from an excess of saturation.

Example 3: Coupling of the Anx FF to the Cells

One method of producing the coupling of Anx FF to erythrocytes is tomix:

30 μl of a 30% suspension of erythrocytes,

30 μl of Anx FF,

6 μl of 125 mM CaCl₂,

7.5 μl of 4% BSA,

256 μl of buffer.

The reaction mixture is incubated for 30 min at 37° C. and then dilutedwith 6 ml of TRIS buffer to which has been added 0.1% BSA and 5 mM ofDTT.

Example 4: Separation of the Cells Retained by Anx FF from theUnretained Cells

This separation is carried out by an automatic apparatus, an example ofwhich, given in FIG. 1 is the following: a tygon tube is placed in amagnetic field and connected to various storage containers via solenoidvalves. The electromagnetic field is brought into action and thereaction mixture described in Example 3 above is, after an incubation at37° C., introduced into the tube by actuating the appropriate valve andpasses through the magnetic field at a rate of 120 ml per hour, and theeffluent is collected.

A valve controlling a washing storage container is actuated and 15 ml ofbuffer pass through the tube. The washing buffer is harvested at thesame level as the effluent consisting of cells not retained by the AnxFF.

The cells retained by the Anx FF are eluted from the tube by switchingoff the magnetic field and by harvesting the cells retained in 8 ml ofbuffer. The cells retained and not retained are concentrated bycentrifuging at 3000 g for 7 min and at 4° C. and then counted beforeany eventual subsequent treatment or characterization. A quantificationof the receptors with annexine in a cell population can be done byemploying annexine tagged with iodine 125.

If reference is made to FIG. 1, during the first stage of washing withphysiological water the valves 1 and 4 are open and the valves 2, 3, 5and 6 are closed; the pump is at its maximum for 2 to 10 minutes andthen stopped.

The buffer is next introduced through the same tube (valves 2 and 4open). The sample of the concentrate is introduced with a flow rate of160 ml/hour, valves 3 and 5 being open (1, 2, 4, 6 closed). The pumpoperates between 3 and 60 seconds and then stopped.

The fraction not retained in the presence of CaCl₂ buffer is nextcollected by passing the buffer at a rate of 160 ml per hour, valves 2and 5 being open (1, 3, 4, 6 closed). The pump is in action for 2 to 10minutes and then turned off.

The fraction retained is obtained after washing with physiologicalwater, valves 1 and 6 being open (2, 3, 4, 5 closed), the electromagnetbeing switched off and the pump brought into action for 2 to 10 minutes.

A last washing with physiological water is performed by opening valves 1and 4 (2, 3, 5, 6 closed) for 2 to 10 minutes.

COMPARATIVE RESULTS

a) Effects of calcium ions.

FIG. 2 shows the percentage of cells retained on the Anx FF particles asa function of the presence or absence of CaCl₂. It is seen that 44% ofthe cells are retained in the presence of calcium ions, whereas only 8 %are retained in the absence of calcium ions.

b) Effect of the age of the erythrocytes,

FIG. 3 shows the percentage of erythrocytes bonded to Anx FF as afunction of the time of in vitro storage of the red corpuscles. It isclear that this percentage increases logarithmically as a function ofthe storage time, indicating an increasing deterioration of themembranes of the said red corpuscles. One week of storage results in 22%of coupling, one month in 30% and three months in 42%.

In the mouse a storage of 24 hours already entails a coupling of 13%,whereas no bonding is observed with freshly collected blood.

c) Effect of the erythrocyte concentration on the separation of theerythrocyte-Anx FF complexes.

This effect is illustrated in FIG. 4, which shows that the resultobtained is independent of the concentration of red corpuscles in thesolution: in the conditions employed, between 20 and 24% of the cellsare retained, whatever the concentration employed.

d) Effect of the various doses of annexine-FFSH on the retention of theerythrocytes.

FIG. 5 shows the percentage of red corpuscles retained on the Anx FF inthe presence of 20 μl or 30 μl of annexine-FFSH. It is seen that 20 μlof Anx FF are sufficient to obtain a maximum retention starting with ayoung erythrocyte population, whereas 30 μl are needed for agederythrocytes.

f) Relationship between the Anx FF coupling of the erythrocytes and thephysiological or pathological state of these same red corpuscles.

The table below shows the results obtained individually on controlsamplings performed by the French Blood Agency.

    ______________________________________                                        FBA     Normal    Sickle cell        Other                                                                          control SR anemia High SR pathologie                                         s                                        ______________________________________                                        19.60   21.73     50.00     36.94    49.76                                      0.00 16.75 49.00 36.37 63.00                                                  14.80 19.56 64.00 51.26 63.00                                                 12.00 27.59 35.00 54.49                                                       11.90 32.00 49.00 49.85                                                       18.00  53.20 46.80                                                            0.00   65.70                                                                  12.60   71.00                                                                 14.00                                                                         5.90                                                                          12.00                                                                         10.00                                                                         8.70                                                                          .sup.( *.sup.) 10.7 ± 5.9 23.5 ± 6.2 50 ± 9.3 51.5 ± 12.3                                            58.6 ± 7.6                            ______________________________________                                         .sup.(*.sup.) Mean                                                       

The mean retention of the red corpuscles on the Anx FF is 10.7 plus orminus 5.9. The results given in the last four columns relate to bloodsamples of subjects who have attended medical analysis laboratories. Thesamples were assigned to us, the anonymity of the subjects beingpreserved. Only the sedimentation rate (SR) was known to us. Resultsgiven in the second column show a moderately raised degree of retentionin subjects with normal SR whose pathology has remained unknown to us.The results given in the fifth column relate to subjects also withnormal SR but exhibiting a markedly higher degree-of retention, in whosecases it was possible to identify the pathology after investigation: onecase of carcinoma (the subject being under chemotherapy), one case ofdeforming rheumatism and one case of onset of rhinopharingitis. Theresults given in the third and fourth columns relate to blood sampleswith a high SR from subjects presenting a known (third column) orunknown pathology (fourth column). These results clearly indicate thatan abnormally high percentage of retention on the Anx FF is thereflection of a physiological or pathological state which must cause thetransfuser to reject the batch of red corpuscles for transfusionpurposes.

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What is claimed is:
 1. A method for obtaining ferrofluid-formingmagnetic particles substituted with dimercaptosuccinic acid (DMSA) whichare capable of being covalently coupled to an effector, the methodcomprising combining DMSA with ferrofluid-forming magnetic particles,thereby producing ferrofluid-forming particles having disulfide bonds(FFSS), and reducing the disulfide bonds of the FFSS at a basic pH toform ferrofluid-forming magnetic particles substituted with DMSA havingreduced thiol groups (FFSH).
 2. The method of claim 1, whereinflocculation of the FFSS takes place, and further comprising(i)centrifuging the flocculated FFSS, and peptizing the flocculated FFSSparticles in a basic solution, and after forming the FFSH, (ii)flocculating the FFSH in an acidic solution, and washing andre-suspending the FFSH in aqueous buffer.
 3. The method of claim 1,further comprising covalently binding an effector to the FFSH to form acovalent effector-FFSH complex wherein the covalent bond is an S--S,C--S, C--C, or C--N bond.
 4. The method of claim 3, further comprisingcovalently attaching a difunctional reactant comprising at least onefunctional group to an effector to produce an effector capable ofcovalently binding to FFSH.
 5. The method of claim 3, furthercomprising, following formation of an effector-FFSH complex, maskingunreacted reactive groups of the FFSH by a saturation treatment withserum albumin.
 6. A composition comprising a ferrofluid-forming magneticparticle complexed with DMSA having reduced thiol groups FFSH.
 7. Thecomposition according to claim 6, wherein the FFSH is covalently coupledto an effector.
 8. The composition according to claim 7, wherein theeffector has a covalently bound difunctional reactant through which itis attached to the FFSH.
 9. The composition of claim 7, wherein theeffector that is coupled to FFSH is an annexine.
 10. The compositionaccording to claim 7 wherein the molar relationship between the FFSH andthe effector is between 1:1 and 3:1.
 11. A method for identifyingmolecules or cells having a ligand that forms an affinity complex withan effector coupled to FFSH, the method comprising:a) mixing themolecules or cells with the effector coupled to FFSH; and b) subjectingthe mixture to a magnetic field to separate the molecules or cellshaving said ligand from the molecules or cells that do not have such aligand.
 12. The method of claim 11, further comprising detecting themolecules or cells which bind to the effector-coupled FFSH particles.13. The method of claim 12, further comprising identifying cellspresenting an abnormal or pathological physiological state associatedwith the presence in cellular outer membranes of an anionic phospholipidthat forms an affinity complex with annexine as the effector coupled toFFSH, the method comprising:a) complexing cells, in the presence ofcalcium ions, with annexine-FFSH; b) subjecting the complex of cells andannexine-FFSH to a magnetic field and washing, so as to separate cellsbound annexine-FFSH from cells not bound to annexine-FFSH; and c)determining the fraction of the cells bound to annexine-FFSH.
 14. Themethod of claim 13 wherein the anionic phospholipid in the cellularouter membranes that is associated with an abnormal or pathologicalstate is phosphatidylserine or phosphatidylethanolamine.
 15. The methodof claim 13 wherein the cells are erythrocytes from an individual, andwherein identification of an abnormally high fraction of saiderythrocytes having said anionic phospholipid in their outermembranes:a) indicates the presence of erythrocytes presenting anabnormal physiological state; and/or b) permits determining andcontrolling the state of health of blood cells in a blood sample beforetransfusion; and/or c) is indicative of a pathological condition in theindividual from whom the erythrocytes were obtained.
 16. The method ofclaim 13 wherein the annexine-FFSH particles have a diameter that isbetween 3 and 30 nm.
 17. The method of claim 13 wherein the calcium ionspresent in step a) are provided as CaCl₂ in a concentration between 0.5and 5 mM.
 18. A device for identifying or separating compounds or cellshaving a ligand that forms an affinity complex with an effector coupledto a ferrofluid-forming magnetic particle substituted with DMSA havingreduced thiol groups according to the method of claim 11, the devicecomprising:a) an electromagnet; b) one or more channels able toselectively direct the flow of sample, buffer, and wash solutionsthrough the magnetic field of the electromagnet to effect separationinto retained and non-retained fractions; c) a pump connected to saidchannels; and d) one or more apertures from which the retained andnon-retained fractions are collected.
 19. A device for identifying orseparating from a sample solution compounds or cells having a ligandthat forms an affinity complex with an effector coupled to aferrofluid-forming magnetic particle substituted with DMSA havingreduced thiol groups according to the method of claim 11, the devicecomprising:a) a means for subjecting the sample solution to a magneticfield; b) a means for directing the sample solution through saidmagnetic field to separate the compounds or cells having the ligand; andc) a means for collecting the compounds or cells having the ligandseparately from compounds or cells not having the ligand.